Aerosol Particle Size Influences the Infectious Dose and Disease Severity in a Golden Syrian Hamster Model of Inhalational COVID-19.

Background: Significant evidence suggests that SARS-CoV-2 can be transmitted via respiratory aerosols, which are known to vary as a function of respiratory activity. Most animal models examine disease presentation following inhalation of small-particle aerosols similar to those generated during quiet breathing or speaking. However, despite evidence that particle size can influence dose-infectivity relationships and disease presentation for other microorganisms, no studies have examined the infectivity of SARS-CoV-2 contained in larger particle aerosols similar to those produced during coughing, singing, or talking. Therefore, the aim of the present study was to assess the influence of aerodynamic diameter on the infectivity and virulence of aerosols containing SARS-CoV-2 in a hamster model of inhalational COVID-19. Methods: Dose-response relationships were assessed for two different aerosol particle size distributions, with mass median aerodynamic diameters (MMADs) of 1.3 and 5.2 µm in groups of Syrian hamsters exposed to aerosols containing SARS-CoV-2. Results: Disease was characterized by viral shedding in oropharyngeal swabs, increased respiratory rate, decreased activity, and decreased weight gain. Aerosol particle size significantly influenced the median doses to induce seroconversion and viral shedding, with both increasing ∼30-fold when the MMAD was increased. In addition, disease presentation was dose-dependent, with seroconversion and viral shedding occurring at lower doses than symptomatic disease characterized by increased respiratory rate and decreased activity. Conclusions: These results suggest that aerosol particle size may be an important factor influencing the risk of COVID-19 transmission and needs to be considered when developing animal models of disease. This result agrees with numerous previous studies with other microorganisms and animal species, suggesting that it would be generally translatable across different species. However, it should be noted that the absolute magnitude of the observed shifts in the median doses obtained with the specific particle sizes utilized herein may not be directly applicable to other species.

Comparison of Dose-Response Relationships for Two Isolates of SARS-CoV-2 in a Nonhuman Primate Model of Inhalational COVID-19.

Background: As the COVID-19 pandemic has progressed, numerous variants of SARS-CoV-2 have arisen, with several displaying increased transmissibility. Methods: The present study compared dose-response relationships and disease presentation in nonhuman primates infected with aerosols containing an isolate of the Gamma variant of SARS-CoV-2 to the results of our previous study with the earlier WA-1 isolate of SARS-CoV-2. Results: Disease in Gamma-infected animals was mild, characterized by dose-dependent fever and oronasal shedding of virus. Differences were observed in shedding in the upper respiratory tract between Gamma- and WA-1-infected animals that have the potential to influence disease transmission. Specifically, the estimated median doses for shedding of viral RNA or infectious virus in nasal swabs were approximately 10-fold lower for the Gamma variant than the WA-1 isolate. Given that the median doses for fever were similar, this suggests that there is a greater difference between the median doses for viral shedding and fever for Gamma than for WA-1 and potentially an increased range of doses for Gamma over which asymptomatic shedding and disease transmission are possible. Conclusions: These results complement those of previous studies, which suggested that differences in exposure dose may help to explain the range of clinical disease presentations observed in individuals with COVID-19, highlighting the importance of public health measures designed to limit exposure dose, such as masking and social distancing. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, as well as to inform dose selection in future studies examining the efficacy of therapeutics and vaccines in animal models of inhalational COVID-19.

Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19.

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID50 (95% CI: 23-363 TCID50), and was significantly lower than the median dose for fever (256 TCID50, 95% CI: 102-603 TCID50), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.

SARS-CoV-2 is rapidly inactivated at high temperature.

In the absence of a vaccine, preventing the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the primary means to reduce the impact of the 2019 coronavirus disease (COVID-19). Multiple studies have reported the presence of SARS-CoV-2 genetic material on surfaces suggesting that fomite transmission of SARS-CoV-2 is feasible. High temperature inactivation of virus has been previously suggested, but not shown. In the present study, we investigated the environmental stability of SARS-CoV-2 in a clinically relevant matrix dried onto stainless steel at a high temperature. The results show that at 54.5 °C, the virus half-life was 10.8 ± 3.0 min and the time for a 90% decrease in infectivity was 35.4 ± 9.0 min. These findings suggest that in instances where the environment can reach temperatures of at least 54.5 °C, such as in vehicle interior cabins when parked in warmer ambient air, that the potential for exposure to infectious virus on surfaces could be decreased substantially in under an hour.

Influence of aerodynamic particle size on botulinum neurotoxin potency in mice.

OBJECTIVE: For many agents, the aerodynamic particle size can affect both the virulence and disease course in animal models. Botulinum neurotoxins (BoNTs), which are widely known as potential bioterrorism agents, have been shown to be toxic via multiple routes of exposure, including small particle inhalation (1-3 µm MMAD). However, the impact of larger particle sizes on the potency of BoNT has not been previously reported. In this study, we compared the potency of BoNT in small and large particle aerosols. MATERIALS AND METHODS: Outbred mice (ICR (CD-1®)) were exposed to BoNT-containing aerosols with differing mass median aerodynamic diameters (MMADs) of 1.1, 4.9, and 7.6 microns. The effects of bioaerosol sampler and inhalation exposure modality were studied. RESULTS AND DISCUSSION: Collecting aerosolized BoNT onto gelatin filters or into liquid impingers resulted in equivalent estimates of aerosol concentration. Nose-only and whole-body inhalation exposure resulted in nearly identical estimates of the median lethal dose (LD50). The LD50 for inhaled BoNT increased approximately 50-fold when the median aerodynamic particle size was increased from 1.1 to 4.9 µm, from 139 (95% CI: 111-185) to 7324 (95% CI: 4287-10 891) mouse intraperitoneal median lethal doses (MIPLD50). These results demonstrate the importance of aerodynamic particle size and regional deposition patterns with regards to BoNT inhalational toxicity. CONCLUSIONS: These data will be useful for medical countermeasure development, as well as biodefense preparedness modeling by demonstrating that the estimates of dose and toxicity of an inhaled aerosol containing BoNT can be significantly affected by a range of factors.

Increasing Temperature and Relative Humidity Accelerates Inactivation of SARS-CoV-2 on Surfaces.

Coronavirus disease 2019 (COVID-19) was first identified in China in late 2019 and is caused by newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous studies had reported the stability of SARS-CoV-2 in cell culture media and deposited onto surfaces under a limited set of environmental conditions. Here, we broadly investigated the effects of relative humidity, temperature, and droplet size on the stability of SARS-CoV-2 in a simulated clinically relevant matrix dried on nonporous surfaces. The results show that SARS-CoV-2 decayed more rapidly when either humidity or temperature was increased but that droplet volume (1 to 50 µl) and surface type (stainless steel, plastic, or nitrile glove) did not significantly impact decay rate. At room temperature (24°C), virus half-life ranged from 6.3 to 18.6 h depending on the relative humidity but was reduced to 1.0 to 8.9 h when the temperature was increased to 35°C. These findings suggest that a potential for fomite transmission may persist for hours to days in indoor environments and have implications for assessment of the risk posed by surface contamination in indoor environments.IMPORTANCE Mitigating the transmission of SARS-CoV-2 in clinical settings and public spaces is critically important to reduce the number of COVID-19 cases while effective vaccines and therapeutics are under development. SARS-CoV-2 transmission is thought to primarily occur through direct person-to-person transfer of infectious respiratory droplets or through aerosol-generating medical procedures. However, contact with contaminated surfaces may also play a significant role. In this context, understanding the factors contributing to SARS-CoV-2 persistence on surfaces will enable a more accurate estimation of the risk of contact transmission and inform mitigation strategies. To this end, we have developed a simple mathematical model that can be used to estimate virus decay on nonporous surfaces under a range of conditions and which may be utilized operationally to identify indoor environments in which the virus is most persistent.

Cigarette smoke induces systemic defects in cystic fibrosis transmembrane conductance regulator function.

RATIONALE: Several extrapulmonary disorders have been linked to cigarette smoking. Smoking is reported to cause cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the airway, and is also associated with pancreatitis, male infertility, and cachexia, features characteristic of cystic fibrosis and suggestive of an etiological role for CFTR. OBJECTIVES: To study the effect of cigarette smoke on extrapulmonary CFTR function. METHODS: Demographics, spirometry, exercise tolerance, symptom questionnaires, CFTR genetics, and sweat chloride analysis were obtained in smokers with and without chronic obstructive pulmonary disease (COPD). CFTR activity was measured by nasal potential difference in mice and by Ussing chamber electrophysiology in vitro. Serum acrolein levels were estimated with mass spectroscopy. MEASUREMENTS AND MAIN RESULTS: Healthy smokers (29.45 ± 13.90 mEq), smokers with COPD (31.89 ± 13.9 mEq), and former smokers with COPD (25.07 ± 10.92 mEq) had elevated sweat chloride levels compared with normal control subjects (14.5 ± 7.77 mEq), indicating reduced CFTR activity in a nonrespiratory organ. Intestinal current measurements also demonstrated a 65% decrease in CFTR function in smokers compared with never smokers. CFTR activity was decreased by 68% in normal human bronchial epithelial cells exposed to plasma from smokers, suggesting that one or more circulating agents could confer CFTR dysfunction. Cigarette smoke-exposed mice had decreased CFTR activity in intestinal epithelium (84.3 and 45%, after 5 and 17 wk, respectively). Acrolein, a component of cigarette smoke, was higher in smokers, blocked CFTR by inhibiting channel gating, and was attenuated by antioxidant N-acetylcysteine, a known scavenger of acrolein. CONCLUSIONS: Smoking causes systemic CFTR dysfunction. Acrolein present in cigarette smoke mediates CFTR defects in extrapulmonary tissues in smokers.

Development of a murine nose-only inhalation model of influenza: comparison of disease caused by instilled and inhaled A/PR/8/34.

Influenza continues to cause widespread disease and death during winter months. In preclinical studies to evaluate the potential efficacy of drugs and vaccines, influenza challenge virus is usually instilled into the noses of animals in the form of large liquid drops. Since inhalation of aerosolized influenza is commonly associated with human transmission, instillation of challenge virus raises uncertainty about the applicability of results. In order to compare the challenge methods, we established conditions to generate influenza aerosols with a mass median aerodynamic diameter (MMAD) of 1 µm that were delivered to mice in a nose-only inhalation system. In this report, we describe the system and compare the 50% lethal dose (LD(50)) of instilled and inhaled A/PR/8/34 (PR8) in BALB/c mice. The estimated LD(50) for inhaled virus was 8.7 plaque forming units (PFU) and the mean time to death was 7.7 days, whereas the estimated LD(50) for instilled virus was 51.6 PFU and the mean time to death was 8.2 days. Our results show that mice are more sensitive to inhaled virus than virus delivered by intranasal instillation. The murine nose-only inhalation model of influenza infection can be used to infect large numbers of animals simultaneously with well-characterized, homogenous PR8 bioaerosol in a controlled and reproducible manner. This model provides the means to evaluate the efficacy of drug and vaccine candidates against the relevant route of challenge, thereby providing data that may better predict clinical outcome.

Antibody responses of variable lymphocyte receptors in the lamprey.

Lamprey and hagfish, the living representatives of jawless vertebrates, use genomic leucine-rich-repeat cassettes for the combinatorial assembly of diverse antigen receptor genes encoding variable lymphocyte receptors of two types: VLRA and VLRB. We describe here the VLRB-bearing lineage of lymphocytes in sea lamprey. These cells responded to repetitive carbohydrate or protein determinants on bacteria or mammalian cells with lymphoblastoid transformation, proliferation and differentiation into plasmacytes that secreted multimeric antigen-specific VLRB antibodies. Lacking a thymus and the ability to respond to soluble protein antigens, lampreys seem to have evolved a B cell-like system for adaptive humoral responses.

Structure and specificity of lamprey monoclonal antibodies.

Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores. The recombinant VLR-B antibodies possess 8-10 uniform subunits that collectively bind antigen with high avidity. Sequence analysis, mutagenesis, and modeling studies show that antigen binding involves residues in the beta-sheets lining the VLR-B concave surface. EM visualization reveals tetrameric and pentameric molecules having a central core and highly flexible pairs of stalk-region "arms" with antigen-binding "hands." Remarkable antigen-binding specificity, avidity, and stability predict that these unusual LRR-based monoclonal antibodies will find many biomedical uses.

The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis.

The outermost layer of the Bacillus anthracis spore is the exosporium, which is composed of a paracrystalline basal layer and an external hair-like nap. The filaments of the nap are formed by a collagen-like glycoprotein called BclA, while the basal layer contains several different proteins. One of the putative basal layer proteins is ExsY. In this study, we constructed a DeltaexsY mutant of B. anthracis, which is devoid of ExsY, and examined the assembly of the exosporium on spores produced by this strain. Our results show that exosporium assembly on DeltaexsY spores is aberrant, with assembly arrested after the formation of a cap-like fragment that covers one end of the forespore-always the end near the middle of the mother cell. The cap contains a normal hair-like nap but an irregular basal layer. The cap is retained on spores prepared on solid medium, even after spore purification, but it is lost from spores prepared in liquid medium. Microscopic inspection of DeltaexsY spores prepared on solid medium revealed a fragile sac-like sublayer of the exosporium basal layer, to which caps were attached. Examination of purified DeltaexsY spores devoid of exosporium showed that they lacked detectable levels of BclA and the basal layer proteins BxpB, BxpC, CotY, and inosine-uridine-preferring nucleoside hydrolase; however, these spores retained half the amount of alanine racemase presumed to be associated with the exosporium of wild-type spores. The DeltaexsY mutation did not affect spore production and germination efficiencies or spore resistance but did influence the course of spore outgrowth.

Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis.

Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T(m)) of 37 degrees C, like mammalian collagen. However, rBclA trimers exhibited T(m) values of 84 degrees C and 95 degrees C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T(m) values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.

Peptide ligands that bind selectively to spores of Bacillus subtilis and closely related species.

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.